Autoantibodies to phospholipids (aPL) occur in certain patients with systemic lupus erythematosus, other systemic autoimmune diseases, and in the primary aPL syndrome. Their presence is associated with an increased risk of thrombosis, recurrent fetal loss, and thrombocytopenia. It is thought that these autoantibodies may exert a direct pathogenic effect by interfering with hemostatic reactions that occur on the surface of platelets and vascular endothelial cells. It has recently been demonstrated that aPL do not react with phospholipid alone, but rather with a complex antigen comprised of phospholipid and the plasma protein beta2-glycoprotein I (beta2GPI). Interestingly, beta2GPI has certain of the key characteristics previously attributed to aPL, i.e., binding to anionic phospholipids and inhibition of the prothrombinase activity. Preliminary data demonstrate that the ability of certain aPL to inhibit prothrombin activation, i.e., lupus anticoagulant activity, is dependent upon the presence of beta2GPI. It is hypothesized that antiphospholipid antibodies exert their pathogenic effects by enhancing the binding of beta2GPI to phospholipids, thereby inhibiting phospholipid-dependent reactions of the hemostatic system. the long-term objective of the proposed research is to define the mechanisms by which aPL may cause a thrombotic diathesis. The proposed studies will focus on the effect of aPL on the interactions of beta2GPI and phospholipids, both in binding studies and hemostatic reactions. The first aim is to characterize quantitatively the binding of beta2GPI to phospholipid membranes in the presence of absence of aPL. Binding of beta2GPI alone will be quantitated by 90o light scattering and fluorescence energy transfer. The effect of aPL on this binding will be determined using nonequilibrium binding assays and fluorescence anisotropy measurements. Additionally, because lupus anticoagulants are reported to be specific for hexagonal (II) phase phospholipids and such structures may play an important role in platelet procoagulant activity, the specificity of beta2GPI and aPL for hexagonal (II) phase lipid will be determined. The second aim is to determine the mechanism by which beta2GPI and aPL inhibit the activation of prothrombin, and incorporate this information into a quantitative model of prothrombin activation. the third aim is to determine the effects of beta2GPI, in the presence or absence of aPL, in phospholipid- dependent reactions of the protein C pathway, i.e., the thrombin/thrombomodulin activation of protein C and the protein C/protein S inactivation of factors Va and VIIIa. Inhibition of the protein C pathway by beta2GPI and aPL may explain the thrombotic diathesis associated with these autoantibodies. It is anticipated that characterization of the interaction of beta2GPI with phospholipid membranes, and the effect of aPL on that interaction, will provide insights into the pathophysiology of the aPL syndrome and suggest novel therapeutic strategies.